Fig. 4From: MLKL regulates Cx43 ubiquitinational degradation and mediates neuronal necroptosis in ipsilateral thalamus after focal cortical infarctionVHL is an E3 ubiquitin ligase for Cx43, and MLKL competes with VHL for binding to Cx43. (A) Co-immunoprecipitation and Western blot to detect Cx43 ubiquitination in SH-SY5Y cells post transfection with Cx43(WT), Cx43(K303G), Ub, and VHL. The ubiquitination level of Cx43 was significantly decreased in VHL + Ub + Cx43 (K303G) transfection group (lane 5) in contrast to VHL + Ub + Cx43 (WT) transfection group (lane 4). (B) Co-immunoprecipitation and Western blot to detect the interaction among Cx43, MLKL, and VHL in SH-SY5Y cells. (C) Representative immunoblots of MLKL, pMLKL, Cx43, and VHL expression in SH-SY5Y cells post TSZ treatment, Cx43, MLKL(WT), and MLKL(S454A) transfection. D, E, F, G) Histogram showing the quantitative analyses of MLKL, pMLKL, Cx43, and VHL protein levels in SH-SY5Y cells after TSZ treatment and plasmids transfection (n ≥ 3 in each group). H) Co-immunoprecipitation and Western blot to detect the interaction among MLKL, pMLKL, VHL, and Cx43 in SH-SY5Y cells after TSZ treatment and plasmids transfection. Each bar represents mean ± standard deviation. *P < 0.05 vs. control group and #P < 0.05 vs. all groups except the control group. MLKL, mixed lineage kinase domain-like protein; Cx43, connexin 43; WT, wild type; MT, mutant; Ub, ubiquitin; VHL, von Hippel-Lindau; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TSZ, TNFα + Smac mimetic + ZVAD-FMKBack to article page